Determination Of Corticosteroids Multi-Residues And Application Of Quantum Dots In Immunoassay

Recently, there has been plenty of attention paid to the occurrence of veterinary drug residues in animal-food products, which can lead to great deal of threat towards human health and environment. Class-selective of multi-residues and combination with new nano-materials to increase the sensitivity had been new trends of immunoassay method, which was a simple, fast and sensitive method for the determination of veterinary drug residues. The study included two sections: immunoassay method for the determination of multi-residues of corticosteroids, the application of quantum dots in immunoassay determination.Immunoassay method for the determination of multi-residues of corticosteroids: three immunogens and coating-antigens were prepared by conjugating derived dexamethasone, betamethasone and flumethasone with bovine serum albumin, ovalbumin respectively. Each immunogen was applied to immunize two New Zealand rabbits to obtain anti-serums, which titers were detected and the better ones were up to 345600,614400,307200. Indirect competitive ELISA methods were developed based on the antibodies with good titers and moreover, the dilution ratio of goat anti-rabbit-HRP, competition time and the pH of PBS solution were optimized. The IC50 of optimized indirect compete ELISA for dexamethasone, betamethasone and flumethasone were 2.66 ng/mL,7.36 ng/mL,6.12 ng/mL respectively. The multi-selectivity of three antibodies were accessed by detecting each cross-reactivity, the result showed that the polyclonal antibody of dexamethasone was best suitable for multi-residues determination, the cross-reactivity towards flumethasone was up to 120 %, betamethasone 73 %, triacinolone 37 %, predinisolone 21 %, but towards several natural glucocorticoids was lower than 0.1 %. The recoveries were detected by spiking four level concentrations (0.375 ng/mL,0.75 ng/mL,1.5 ng/mL,3.0 ng/mL) of DEX in chicken muscle tissue, of which results indicated that the recoveries were between 61 % and 80 % and the coefficients of variation were lower than 15 %. The ELISA method and LC-MS method were applied to analyze the real samples, and it was showed that there was high correlation between the two methods.Application of quantum dot in immunoassay method: Biotinylated denatured bovine serum albumin (Bt-dBSA)-coated cadmium telluride (CdTe) quantum dots (QDs) conjugates were prepared and applied to and simplified the multiplexed floroimmunossay. In this work, the BSA, which was denatured and biotinylated with amine-reactive NHS biotin, was used to coat the QDs, while the biotinylated antibodies were prepared. Immune complex formation was formed using the avidin as bridge to link the Bt-dBSA-QDs to the antibodies. Quantitative determination of five representative chemical drugs (Medroxyprogesterone, Dexamethasone, Gentamicin, Clonazepam and Ceftiofur) was performed by using the QDs with different emission properties. The described QD-based protocols are less time-consuming than the classical one and flexible to expand to other systems for the simultaneous multicolor detection of chemical residues. The LODs of FLISA method for Dexamethasone, Gentamicin, Clonazepam, Medroxyprogesterone and Ceftiofur were 0.32 ng/mL,0.20 ng/mL,0.11 ng/mL,0.14 ng/mL,0.24 ng/mL respectively. The FLISA method for the determination of muilti-residues was developed.